international Journal of Systematic Bacteriology (1 999), 49,3 19-327 Printed in Great Britain

Zygosaccharomyces lentus sp. nov., a new member of the genus Zygosaccharomyces Barker

Hazel Steels,' Christopher J. Bond,* Matthew D. C~llins,~t Ian N. Roberts,' Malcolm Stratfordl and Stephen A. James2

Author for correspondence: Stephen A. James. Tel: +44 1603 255274. Fax: +44 1603 458414. e-mail: [email protected]

1 Microbiology Section, Unusual growth characteristics of a spoilage yeast, originally isolated from Un i I ever Research, spoiled whole-orange drink and previously identified as Zygosaccharomyces Colworth House, Sharnbrook, Bedford bailii, prompted careful re-examination of its taxonomic position. Small- MK44 lLQ, UK subunit rRNA gene sequences were determined for this strain and for four * National Collection of other strains also originally described as 2. bailii but which, in contrast to Yeast Cultures, other strains of this species, grew poorly or not at all under aerobic conditions Department of Genetics with agitation, failed to grow in the presence of 1YO acetic acid and failed to and Microbiology, Institute of Food Research, Norwich grow at 30 "C. Comparative sequence analysis revealed that these strains Laboratory, Nowich represented a phylogenetically distinct taxon closely related to, but distinct NR4 7UA, UK from, Z. bailii and Zygosaccharomyces bisponrs. Furthermore, sequence 3 Department of analysis of the internal transcribed spacer (ITS) region showed that, while all Microbiology, Institute of five strains had identical ITS2 sequences, they could be subdivided into two Food Research, Reading Laboratory, Reading groups based on ITS1 sequences. Despite such minor inter-strain sequence RG6 6B2, UK variation, these could readily be distinguished from all other currently described Zygosaccharomyces species by using ITS sequences. On the basis of the phylogenetic results presented, a new species comprising the five strains, Zygosaccharomyces lentus sp. nov., is described and supporting physiological data are discussed, including a demonstration that growth of this species is particularly sensitive to the presence of oxygen. The type strain of Z. lentus is NCYC D2627T.

~~ ~ Keywords : yeast, Zygosaccharomyces lentus, preservative resistance, spoilage

INTRODUCTION excess of those normally, or legally, encountered. Foods particularly at risk are acidic, pH 2-5-50, Zygosaccharomyces is a yeast genus often regarded as containing high concentrations of fermentable synonymous with food spoilage. General character- (Tilbury, 1980; Thomas & Davenport, 1985). These istics of Zygosaccharomyces yeasts are fermentation of include fruit juices, soft drinks, juice concentrates and sugars, osmotolerance, resistance to preservatives in- syrups, candied fruit, jams and preserves, cream cluding sulphite, sorbic acid and ethanol, formation of eggs, honey, tomato sauce and wines. Spoilage results heat-resistant ascospores and fructophily (preference in yeast clouds, particulates, taints and off-flavours, for fructose). Zygosaccharomyces includes some of the odours and excessive gas production. Yeast fermen- most osmotolerant organisms known, yeasts able to tation can generate CO, up to 6 bar over-pressure, resist concentrations of food preservatives vastly in capable of distorting packaging, causing bottles to

...... ,...... explode and rupturing cans and kegs. Though the t Present address: Dept of Food Science and Technology, Earley Gate, Zygosaccharomyces are not human pathogens prod- University of Reading, Reading RG6 6B2, UK. uct spoilage by these yeasts can result in serioushjury, Abbreviation: ITS, internal transcribed spacer. particularly to the eye, caused by exploding glass The EMBL accession numbers for the sequences reported in this paper are bottles (Grinbaum et al., 1994). Y16814 (185 rDNA of NCYC D26273, Y16815 (ITS1 region of NCYC D26273, Y16816 (ITS2 region of NCYC D26273 and Y16817 (ITS1 region of NCYC The genus Zygosaccharomyces was introduced in 1901 2406). by Barker (van der Walt & Johannsen, 1975) for yeast

00878 0 1999 IUMS 319 H. Steels and others species in which asci resulted from the conjugation of 1994). 2. mellis, like Z. rouxii, is very osmotolerant two individual cells and in which the ascospores were and is frequently isolated from honey (Munitis et al., smooth. The genus Zygosaccharomyces is closely 1976; Farris et al., 1981). 2.mellis is also unable to use related to that of the brewing/baking yeasts, Sac- sucrose as a substrate, unless this sugar has been charomyces. Indeed, for many years Zygosaccharo- chemically inverted by low pH (Scarr & Rose, 1966). myces yeasts were classified under and Here Zygosaccharomyces lentus sp. nov., a new species misnomers such as Saccharomyces rouxii still persist in of Zygosaccharomyces that is heavily implicated in the the literature. Recent authorities (Yarrow, 1984; spoilage of foods is reported. It is genealogically closely Barnett et al., 1990) have reinstated Zygosaccharo- related to Z. bailii, Z. bisporus, Z. rouxii and Z. mellis myces as a separate genus, though small-subunit rRNA but is physiologically distinct, and it appears probable sequencing has since revealed that these yeasts are that strains of this new species have previously been phylogenetically intermixed with species of the genera misidentified as Z. bailii. Kluyveromyces, Saccharomyces and Torulaspora (James et al., 1994, 1996; Cai et al., 1996). However, METHODS despite this phylogenetic heterogeneity, results have shown that , Zygosaccharo- Yeast strains. The yeast strains examined in this study are myces bisporus, Zygosaccharomyces mellis and Zygo- listed in Table 1 and are available from the National saccharomyces rouxii, the four most noted spoilage Collection of Yeast Cultures (NCYC), Norwich, UK, and the Portuguese Yeast Culture Collection (IGC), Oeiras, species, are not only closely related to one another, but Portugal. 2.bisporus strain BV7 was obtained from Unilever also form a distinct (bootstrap value, 100%) genea- Research. Saccharomyces cerevisiae X 1280-1 B is a com- logical lineage (James et al., 1997) worthy of separate monly used haploid strain, widely regarded as a typical wild- generic status. type, and was obtained from the Yeast Genetic Stock Center (Berkeley, CA, USA). All strains were grown on YM agar 2. bailii is a yeast renowned for its exceptional (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 1 YO resistance to preservatives. Ingram (1 960) reported glucose, 2 YOagar; pH 5.5) at 24 "C and maintained on agar growth in 500 p.p.m. benzoic acid at pH < 3.0. Similar slopes at 4 "C. resistance to sorbic acid and sulphite has been recorded Growth conditions & (Pitt, 1974; Hammond Carr, 1976; Neves et al., Starter cultures were grown in 10 ml YEPD broth (1 Ohyeast 1994). Growth in preservatives causes adaptation of Z. extract, 2 YObacteriological peptone, 2 YOglucose; pH 4-0)in bailii, the acquired tolerance of extreme levels of 30 ml capped MacCartney bottles for 48 h at 26 "C. Ex- preservatives, possibly as a result of an induced perimental cultures were inoculated with 1 mg dry cells 1-1 preservative pump (Warth, 1977, 1989). 2.bailii is also (2 x lo4cells ml-l). Shaking cultures comprised 50 ml YEPD remarkable in that it has been reported to cause in 125 ml conical flasks, shaken on an Infors orbital shaker, spoilage from an inoculum of one cell per litre or per 25 mm radius, at 140 r.p.m. at 26 "C. Static cultures com- package (Pitt & Hocking, 1985; Davenport, 1996). 2. prised either 50 ml cultures in conical flasks or 10 ml cultures bailii in capped 30 ml MacCartney bottles, incubated without is osmotolerant, growing well in fruit-juice movement. concentrates, and its natural habitat may be in shrivelled, mummified fruits in orchards (Davenport, Modified atmospheres. Where indicated, cultures were grown 2.bisporus (shaken or static) in atmospheres of air, nitrogen or oxygen. 1975). Genealogically, is the closest relative This was achieved by placing inoculated flasks in gas jars, of 2. bailii (James et al., 1994) and as a result shares normally used for anaerobic plate incubation. Nitrqgen or many of its characteristics. However, Z. bisporus is oxygen was flushed through the jars for 2 min before they marginally more osmotolerant and appears less re- were sealed. Jars containing flasks were then placed on sistant to preservatives (Tilbury, 1980) and 2. bisporus orbital shakers or incubated without movement. is unable to grow on sucrose (Barnett et al., 1990), Growth in acetic acid. Ability of yeast strains to grow on media whereas 2. bailii uses this substrate, albeit poorly. Z. containing 1 YOacetic acid was determined by using MAC rouxii is also renowned for its extreme osmotolerance, agar plus 1 YOacetic acid (1 YOmannitol, 1 YOyeast extract, being found at a water activity of 0.62 in fructose and 2 % agar and glacial acetic acid, added after autoclaving, at 0.65 in glucose/glycerol (Tilbury, 1980). As a conse- 1 YO,v/v). Growth on this medium is a characteristic of 2. quence, Z. rouxii is particularly noted for its spoilage bailii (Davenport, 1981). of sugar syrups and juice concentrates. This yeast Phenotypic characterization. Yeast strains were charac- cannot ferment sucrose but, like 2. bailii, displays a terized phenotypically by using the API ID 32C strip system high degree of resistance to preservatives. In contrast, (bioM6rieux)for quick identification and standard methods 2. mellis has less frequently been reported to cause for complete yeast identification (van der Walt & Yarrow, spoilage (Sand & van Grimsven, 1976), though this is 1984). perhaps due more to the fact that, until recently, this PCR amplification of the 185 rDNA and internal transcribed yeast was regarded as a synonym of 2. rouxii (Barnett spacer (ITS) region. Amplification of the 18s rRNA gene was et al., performed as described by James et al. (1 994). The entire ITS 1990). Indeed, it was only through a nuclear (n) region was amplified with primers P3490 and pITS4 as DNA-nDNA hybridization study of the Zygosacchar- previously described by James et al. (1998). The amplified omyces that Kurtzman (1990) established this yeast as products were purified by using a QIAquick PCR puri- a genetically distinct species, a finding later confirmed fication kit (Qiagen) according to the manufacturer's in- by small-subunit rRNA sequencing (James et al., structions.

~ 320 International Journal of Systematic Bacteriology 49 Zygosaccharomyces lentus sp. nov.

Table 7. Yeast strains used in this study

I Species Strain Source of isolate Saccharomyces cerevisiae X2 180-1B Unknown Zygosaccharomyces bailii NCYC 417 Unknown NCYC 563 Sorghum brandy NCYC 1416T Unknown NCYC 1427 Unknown NCYC 1766 Spoiled grape and blackcurrant juice Zygosaccharomyces bisporus BV7 Middle-Eastern soft drink spoilage NCYC 1495T Unknown Zygosaccharomyces lentus IGC 5207 Spoiled orange beverage, UK IGC 5316 Wine, France NCYC 1601 Orange squash drink NCYC 2406 Spoiled tomato ketchup, UK NCYC D2627T Spoiled whole-orange juice, UK

Sequence determination and analysis. Direct sequencing of both the 18s rRNA gene and ITS PCR products was performed by using a Taq DyeDeoxy terminator cycle- sequencing kit (ABI) and an Omnigene thermal cycler (Hybaid) according to the manufacturers' recommend- ations. Complete 18s rDNA and ITS sequences were determined by using primers described previously by James et al. (1994,1996), with the exception that primers WIL1 and WIL2 (James et al., 1998) were used for 18s rDNA sequencing in preference to primers M760 and P760 (James et al., 1994), as they generated higher quality template for subsequent sequence analysis. Purified sequence reaction mixtures were electrophoresed with an Applied Biosystems model 373A automatic DNA sequencer. Analysis of sequence data. The 18s rRNA gene sequences were aligned by using the multiple-sequence alignment program PILEUP (Feng & Doolittle, 1987) contained within the GCG software package (Genetics Computer Group, 1991) version 8.1. Alignments were adjusted manually. 18s rRNA gene sequence identity values were calculated by using the program GAP. Phylogenetic analyses were per- formed by using PHYLIP (phylogeny inference package ; Felsenstein, 1993) version 3.572. A distance matrix was obtained by using the DNADIST program and an unrooted phylogenetic tree was constructed by using the neighbour- joining method (Saitou & Nei, 1987) and the NEIGHBOR program. The stability of the individual branches of the tree was assessed by using the bootstrap method (Felsenstein, 1985) with the SEQBOOT,DNADIST, NEIGHBOR and CONSENSE programs. The ITS1 and ITS2 sequences for the five strains of the novel Zygosaccharomyces species were aligned and compared with those of 2. bailii and 2. bisporus. The resulting alignments were adjusted manually. Fig- 7. Photomicrograph of NCYC D2627T grown on malt extract agar for 10d at 22°C. Many cells have formed distinctive RESULTS AND DISCUSSION conjugation tubes, while other cells contain two to four smooth ascospores. Bar, 10 pm. Strain NCYC D2627T was originally isolated in the UK in the mid-1980s from a spoiled whole-orange drink. It was provisionally identified as a strain of 2. bailii on the basis of utilization of carbohydrates as jugation tubes (referred to by some authors as growth substrate, as demonstrated by the API ID 32C ' schmoos ' : Lemontt & MacKay, 1977 ; Drubin, 1991) strip system. Furthermore, growth of this strain on and subsequent formation of one to four smooth malt extract or on cornmeal agar at 24 "C for 1-2 ascospores (Fig. 1). This morphology is also typical of weeks resulted in the production of distinctive con- 2. bailii (Thomas & Davenport, 1985).

International Journal of Systematic Bacteriology 49 32 1 H. Steels and others

4000 I I

n 500 - n r -I 400 -

UE 300 - .- aJ 200 F - n 100 -

Yeast strain - I 02 Air N2 Atmosphere Fig. 2. Lack of growth of NCYC D2627T, NCYC 1601, NCYC 2406, IGC 5207 and IGC 5316 in shaking culture at 140r.p.m. on a 25mm orbital radius shaker, measured after 2d at 26°C in Fig- 4. Growth of NCYC D2627T in YEPD medium under YEPD medium. Other strains shown are Z, bailii NCYC 563 and modified atmospheres. Cultures (50 ml in 125 ml conical flasks) NCYC 1766, S. cerevisiae X2180-1B and Z. bisporus BV7. were placed in gas jars, which were then flushed with nitrogen, oxygen or air for 2min. Jars were then sealed and incubated with shaking (140 r.p.m., 25 mm orbital radius; open bars) or without (static; filled bars). Growth was measured after 7 d at 26 "C.

much slower growth rate. A further anomaly was observed when cultures were incubated in shaking conical flasks. Growth of typical 2. bailii strains was enhanced by shaking, whereas no growth was observed with shaken cultures of strain NCYC D2627T (Fig. 2). g 1000 2. bisporus and S. cerevisiae also grew well in shaking cultures (Fig. 2), as did 2. rouxii (data not shown). Growth characteristics of both static and shaking cultures in YEPD broth at pH 4.0 were investigated in l--dkcI20 40 60 80 greater detail, as shown in Fig. 3. Growth of 2. bailii Time (h) NCYC 563 was shown to be significantly better in

400 r I shaking culture than in static culture. Growth of 2. bailii was complete and reached stationary phase

n within 90 h, but slower growth, with a doubling time of 7 -I 4 h, was observed in the static culture, compared to

WF 2-5 h when the culture was shaken. Final yield (Fig. 3a) was also considerably greater in the shaking culture, with three times more cells being produced compared to the static culture. Fig. 3(b) demonstrates a diametrically opposite re- sponse to agitation in cultures of NCYC D2627T.No growth was obtained with shaking, even after 200 h 50 100 150 200 incubation. Growth in static culture was steady but Time (h) slow, with the doubling time estimated to be 15 h. The final yield was also low compared to 2. bailii NCYC 563, at only 380 mg dry weight cells 1-l. Fig. 3. Growth of (a) Z. bailii NCYC 563 and (b) NCYC D2627T in 50 ml YEPD medium in a 125 ml conical flask at 26 "C. Growth in shaking (B) (140 r.p.m., 25 mm orbital radius) and static (e) Growth in modified atmospheres cultures is shown. Growth was measured as OD,,, and converted to dry weight by using a calibration graph. The lack of growth of NCYC D2627T in shaking culture was investigated briefly. Growth inhibition was likely to be due either to a greater sensitivity of this Growth in shaking and static cultures yeast to oxygen or to the physical agitation of shaking. This was examined by growing cells in both shaking It was observed that, when cultured, strain NCYC and static conditions under an atmosphere of nitrogen, D2627T required a much longer period than 2. bailii to oxygen or air (Fig. 4). In shaking cultures, growth was reach a visible level of growth and appeared to have a only obtained in flasks under an atmosphere of

322 International Journal of Systematic Bacteriology 49 Zygosaccharomyces lentus sp. nov.

Saccharomycesmartiniae CBS 6334 Kluyveromycesyarrowii CBS 8242 Kluyveromycespolysporus NCYC 523 - 99 Saccharomycesexiguus NCYC 8 14

Saccharornyceskunashirensis CBS 7662 - Kluyveromyceslodderae NCYC 14 1 7

...... I ...... Fig. 5. Dendrogram showing the phylogenetic relationship of Z. lentus sp. nov. to type strains of other species of the genera Kluyveromyces, Candida, Saccharomyces, Torulaspora and Zygosaccharomyces, based on 185 rRNA gene sequences. The tree was constructed by using the neighbour-joining method (Saitou & Neil 1987). Bootstrap values, expressed as percentages of 200 replications, are given at branch points (only values >50% are shown). Bar, one estimated base substitution per 1000 nucleotide positions. nitrogen, with no growth observed under air or oxygen. and IGC 5316 in shaking culture only occurred at Static culture resulted in growth under air or nitrogen temperatures close to the upper limit for growth and but, again, not in oxygen. Growth of NCYC D2627T that these strains grew well in shaking culture at in shaking culture under nitrogen demonstrates that temperatures of < 24 "C (data not shown). Therefore, the physical agitation of shaking did not affect growth. these strains cannot be regarded as microaerophiles. Inhibition of growth of NCYC D2627Tin static culture We speculate that these strains are sensitive to oxi- under oxygen shows this strain to be sensitive to dative damage when stressed by temperatures close to oxygen. The higher levels of oxygen available in a the upper growth limit. shaking culture are therefore likely to inhibit growth. However, results suggest that a minimum level of oxygen is required for optimal growth, as the final Growth in 1 % acetic acid yield of cells in a static culture was higher in air than in 2. bailii is a preservative-resistant yeast able to grow in nitrogen. Lack of growth in shaking culture was also the presence of 1 % acetic acid, a fact used as a observed in a further four strains supposedly belonging diagnostic test for this species (Barnett et al., 1990). to 2. bailii, NCYC 1601, NCYC 2406, IGC 5207 and Growth tests on MAC agar plus 1% acetic acid IGC 53 16, all derived from spoiled fruit juice-derived showed growth of 2. bailii strains NCYC 563 and foods. NCYC 1766 and 2.bisporus BV7 but not of any of the Further work showed that the lack of growth of strains five strains unable to grow in shaking culture, NCYC NCYC 1601, NCYC 2406, NCYC D2627T, IGC 5207 D2627T, NCYC 1601, NCYC 2406, IGC 5207 and

International Journal of Systematic Bacteriology 49 323 H. Steels and others

50 Zygosaccharomyces, Torulaspora and Saccharomyces 18s rDNA 2627 AGGATCATTATAGAAAATAATCTGATTGAAGAAGTC--TGAGGGAGCATAC--CAAAGGG species (James et al., 1994, 1996, 1997). 2406 ...... -- -- 1416 TT ...... C..-...--..GA.T..TTGTGTT.TC---- 1427 ...... ATT ...... C..-,..--..GA.C..TTGTGTT.TT---- 417 ...... A.T ...... C..-...--.A.A.C.TTTGTGTT.TT---- 185 rRNA gene sequence analysis 1495 ...... A.T- ...... GCA .-...AA..G..C..T.G.GTTTTTC..C The 18s rRNA gene sequences of strains IGC 5207, 100 IGC 5316, NCYC 1601, NCYC 2406 and NCYC 2627 ACTGTTGTCT-CTGCCGGGCCTGCGCTTAGTTGCGCGGTCTAGTGGGG-GAGTGGTTCT- D2627T were amplified in vitro by PCR and their 2406 ...... -...... -...... - 1416 T .....T-T T.AA...... A...... A.C..A.G.A.T.-- AA nucleotide sequences were determined directly. These 1427 T .....T-T .--- TTAA...... A...... A.C..A.G.A.T.---- 417 T .....CAT------AA...... A...... A.C..A.G.A.T.. TAA five newly determined sequences were aligned with 1495 T ..T.CT.T.G ..TTT ...... A.TA.A.G.A.C.... A complete or near-complete 18s rRNA gene sequences 150 of all species of the genus Zygosaccharomyces, as well as representative members of the genera Kluy- 2627 --GCTCTTTTCAGCTT----TTACAATTAACACACAGTGGAGTTTCTACT---TTTCTTC 2406 -- ...... ------veromyces, Saccharomyces and Torulaspora. Levels of 1416 A--.ATAG ..--A ..CTGGC ..T.....T...... TTT...A... 1427 AA-.ATAG ..--G ...TGGC ..T.....T...... TTT...A... sequence identity were calculated (data not shown) 417 AA-. A.AA GT ..TGGC ..T .....T...... TTT...A... and the distances derived were used to infer phylo- 1495 C------CGGC ...... T ...... ATT ...... genetic relationships. Fig. 5 shows an unrooted tree 200 constructed by using the neighbour-joining method 2627 TTCTTTGGGAT--TGGGCTCCGGCCCTGTCCCAGAGGTAACAATTTTTTT- and depicts the phylogenetic relationship between 2406 ...... -- _- 1416 ...... GGA ....T-T..T. ..AC...... AA ...... T strains IGC 5207, IGC 5316, NCYC 1601, NCYC 1427 ...... GGA.,..T-T..T...GC.T...... -_ T 2406 and NCYC D2627* and other members of the 417 ...... GGA ....T-T..T ..TAC...... _- T 1495 ..TAGGA ....GA ..AAAAT.TAT.TAC ...... -- A.....A genus Zygosaccharomyces and close relatives. Com-

250 parative analysis of the 18s rRNA gene sequence data revealed that strains IGC 5207, IGC 5316, NCYC 2627 -----ATTTTATTTTAAAA------AGTCAAACAA 2406 ----- ...... ------...... c...... ---.... 1601, NCYC 2406 and NCYC D2627T had identical 1416 TTTTTT T.TTACA------C ...... A.CG..T..TAA.... sequences, which displayed high levels of sequence 1427 ATTTT ...... TT.TTATAATAATAATAATAC ...... A-CG ..T..TAA..C. 417 TTTTT------...C ..T.TTATAA------AATAC ...... A-CG..T..TAA... C identity with the other Zygosaccharomyces species 1495 TTTT---- ...... C.--CACAGTCAAATGAATAC.-----.A.------AA.... (ranging from 98.1 to 99.8 YO).However, despite such 300 values, the 18s rRNA gene sequences of these strains . 5.85 rDNA 2627 ACAAAATATTCAIAACTTTCIA were not identical to the 2. bailii type strain (NCYC 2406 ...... 1416T), exhibiting five base differences. As Fig. 5 1416 ...... 1427 .-...... shows, these strains are closely related phylogenetically 417 .- ...... 2. bailii 2. bisporus 1495 TT.T...... to both and but are clearly representatives of a distinct Zygosaccharomyces species. Indeed, a detailed analysis of the sequences Fig. 6. Aligned ITS1 sequences of strains NCYC D2627T (2627) and NCYC 2406 (2406), Z. bailii strains NCYC 417 (417), NCYC revealed that strains IGC 5207, IGC 5316, NCYC 1416T (1416) and NCYC 1427 (1427), and Z. bisporus strain NCYC 1601, NCYC 2406 and NCYC D2627T could be 1495T (1495). Dots indicate identity, dashes indicate gaps distinguished readily from 2. bailii, its genealogically introduced to maximize alignment. close relative 2. bisporus (James et al., 1994) and all other Zygosaccharomyces species by the possession of a distinctive seven-base signature sequence (TTTT- IGC 5316. This shows not only that these yeasts are ACT) at positions 678-682 (S. cerevisiae numbering; more sensitive to acetic acid but that they are readily Mankin et al., 1986) in the V4 region of the gene (Neefs distinguished from 2. bailii strains by this test. et al., 1991). In comparison, 2. bailii has an eight-base sequence (TTTTAATT), while 2. bisporus has a nine- base sequence (TTTTAATTT), at these positions Maximum growth temperature (James et al., 1994). A further physiological distinction between the five strains and 2. bailii concerned the temperature range ITS sequence analysis for growth. 2. bailii is reported to be able to grow at The nucleotide sequences of the ITSl and ITS2 region 30 "C, with strain-dependent growth at 35 and 37 "C Zygosaccharomyces (Barnett et al., 1990). However, strains NCYC for all five strains of the novel species were determined by sequencing PCR-amplified D2627T, NCYC 1601, NCYC 2406, IGC 5207 and 2. bailii, IGC 5316 did not grow, even in static culture, at 30 "C fragments directly. Unlike where size vari- or above. ation in both ITSl and ITS2 was found between different strains (James et al., 1996), no such size Slow growth rate, lack of growth in shaking culture, polymorphisms were observed between strains IGC inhibition of growth by 1 YOacetic acid and inability to 5207, IGC 5316, NCYC 1601, NCYC 2406 andNCYC grow at 30 "C all suggested that the taxonomic status D2627T. The ITSl and ITS2 regions were 255 and of these five strains should be re-examined by using 227 bp, respectively, and as such are smaller than their 18s rRNA gene sequencing, as previously applied to counterparts in both 2.bailii (ITS I, ranging from 266

324 In ternationa I lo urnaI of Systematic Bacteriology 49 Zygosaccharomyces lentus sp. nov.

5.8s rDNA 2627 AGCGTCATTTCCTTCTCAAACATTCGTGTTTGGTGGTGAGTGATACTCTGTGTAACAGCT sequence data presented that strains IGC 5207, IGC 1416 ...... A ...... T.T-TCA T. 5316, NCYC 1601, NCYC 2406 and NCYC D2627T 1427 ...... A ...... T.T.TTA T. 417 ...... A ...... A...... T.-.T.T T. have been incorrectly identified as Z. bailii strains. The 1495 ...... C.T ...... A ...... TA..AC--- ITSl and ITS2 sequence data are in good agreement 100 with both the 18s rRNA gene sequence and the

2627 TGAGTTAGCTTGAAATTGTAAGCCTTTTGGGT---GCGT-TGGCA-AGATGTTTTGGGCG physiological data (Fig. 5) and clearly identify these 1416 ..G ....A...... C...... GAAC....G...-GT-GA-.....A.... five strains as belonging to a hitherto unknown species 1427 ..G ....A...... C...... GA-C....G...-GT-GA-.....A.... 417 ..G ....A...... C...... GAT-....G.C.-GT.GA-.....A.... of the genus Zygosaccharomyces. 1495 ..G ...... C...... GAT-....C.A.AGA.GA-.....A....

150 Latin diagnosis of Zygosaccharomyces lentus sp. nov. 2627 GAGACGCCCAGG-CAGCCG--ATTTTCTTT--ACCAAATGTCGTATTAGGTTTTACCGAC 1416 ..A ... T.TT.CT .-T..TCT---...C.--A...... 1427 ..A ...T.TT.CT .-T..TCT---...C.--A...... Cultura in agaro ' morphologica ' (Difco) post 48 horas 417 ..A ...T.TT.CT .-T..TTT--- ...C.--A...... ad 24 "C : cellulae globosae vel ovoideae (3.0-8.0 x 3.0- 1495 ..A ...T.TG.CT.CT ..TCT--- ...... 11.0 pm), singulae vel binae, adhaerentes, per gem- 200 mationem multipolarem reproducentes. In agaro farina 2621 TC-GGCAGACGG-----TGGAG------CGGACTGTG------Zeae maydis vel tuberibus Solani tuberosi concocto 1416 ..C.A ...... GACTA- ....ATTG-GGTGAGTGATAGCATAT..AG..C..CCTAA- 1427 ..C.A ...... GACTA- ....ATTG-GGTGAGTGATAGCATAT..AG..C..CCTAA- pseudomycelium primitivum adest. Asci ovoidei per 417 ..C.A ...... GACTA- ....ATTG-GGTGAGTGAGTGATAGCAATAT..AG..C..CTTAAA conjugationem cellularum vegetativarum oriuntur, asco- 1495 ..-...... A.GTTGC .....ACTGAGGTGGGTGATAGATAT..A,..------sporas ovales continentes, ex ascis non liberantur. Post 250 7-14 dies ad 24 "C in agaro farinae Zeae maydis 2627 ------_-_-----_TTCATTTTCCTTGTGCGCCTT-GGCAAACAATGTTCTCAAAGC formantur. Glucosum et sucrosurn fermentantur at non 1416 -----_------___TT ....TT ...C ...... G...... AC...... T 1427 ------_--_----___TT .... TT ...C ...... G...... AC...... T galac t osum, malt osum, cellob iosum, t rehalosum, lact o- 417 AGAAAAAAGATCTCTTT ..TT ....TT ...C ...... G...... AC...... T sum, melibiosum, rafinosum, melezitosum, inulinum nec 1495 -GAAAAAAG------.TTC ..C...... T...... AC....T.... amylum. Glucosum, alcohol aethylicum, gly cerinurn, D-

265 rDNA glueitohm, glucono-D-lactonum,ethylaminumet cada- 2627 TTGACCTCAA verinum assimilantur at non galactosum, L-sorbosum, 1416 ...... 1427 ...... sucrosum, maltosum, cellobiosurn, trehalosum, lacto- 417 ...... sum, melib iosum, rafinosum, melezit osum, inulinurn, 1495 ...... amylum, xylosum, L-arabinosum, D-arabinosum, D- r ibosum, L-rhamnosum , ery thr it ohm, r ibitolum, galac- Fig. 7. Aligned ITS2 sequences of NCYC D2627T (2627), Z. bailii strains NCYC 417 (417), NCYC 1416T (1416) and NCYC 1427 titohm, D-mannitolum, methylurn a-D-ghcosidum, sak- (1427), and Z. bisporus strain NCYC 1495T (1495). Dots indicate cinum, acidum lacticum, acidum succinicum, acidum identity, dashes indicate gaps introduced to maximize citricum, inositolum, D-glucosaminum, metanolum, alignment. xylitolum nec nitras kalicus. Crescit in medio cum 50 YO glucoso. Non crescit in medio 0.0 1 % cycloheximido addito, neque in medio 1 YOacido acetic0 addito, neque in medio vitaminis egente. Typus depositus in collectionis to 281 bp; ITS2, ranging from 258 to 278 bp) and Z. 'National Collection of Yeast Cultures ', Norwich, bisporus (ITS 1,269 bp ; ITS2,25 1 bp) strains (James et Britannia (NCYC D2627T). al., 1996). Description of Zygosaccharomyces lentus sp. nov. The ITSl and ITS2 sequences of the five strains of the novel Zygosaccharornyces species were aligned with Zygosaccharomyces lentus (len'tus. L. adj. lentus slow, those of 2. bailii strains NCYC 417, NCYC 1416Tand apathetic, referring to the slow growth of this yeast). NCYC 1427 and Z. bisporus strain NCYC 1495Tand these alignments are shown in Figs 6 (ITS1) and 7 On morphology agar, after 48 h growth at 24 "C, the (ITS2). As Fig. 6 shows, the five strains of the novel cells are spherical to ovoid (3.0-8-0 x 3.0-1 1.0 pm) and Zygosaccharomyces species could be subdivided into occur singly, in pairs or in groups. Budding is two groups, based on a single base substitution (C to multipolar. Ill-formed pseudohyphae are observed in A) in their ITSl sequences. The first group comprised cultures grown on corn meal agar and potato agar. strains IGC 5207, IGC 5316, NCYC 1601 and NCYC Oval asci containing two to four round-to-oval asco- D2627T, while the second group comprised strain spores are formed after incubation for 1 to 2 weeks at NCYC 2406 only. In contrast, all five strains possess 24 "C on corn meal agar (Fig. 1). Asci are persistent. identical ITS2 sequences (Fig. 7). Conjugation occurs between separate cells. Ferments glucose and sucrose. Does not ferment galactose, As Figs 6 and 7 show, the five strains can be ma1t ose, cellobiose, trehalose, lactose, melibiose, distinguished readily from Z. bailii and Z. bisporus raffinose, melezitose, inulin or starch. Assimilates strains on the basis of their distinctive ITSl and ITS2 glucose, ethanol, glycerol, D-glucitol, D-glucono-13- sequences. To date, three different ITS sequence lactone, ethylamine hydrogen chloride and cadaverine. variants have been identified from 2. bailii strains Does not assimilate galactose, L-sorbose, sucrose, (James et al., 1996). However, it is quite clear from the maltose, cellobiose, trehalose, lactose, melibiose, raf-

International Journal of Systematic Bacteriology 49 325 H. Steels and others finose, melezit o se, inulin, starch, xylo se, L- arabino se, etative Microbes, pp. 89-1 10. Edited by F. A. Skinner & W. B. D-arabinose, D-ribose, L-rhamnose, erythritol, ribitol, Hugo. London: Academic Press. galactitol, D-mannitol, methyl a-D-glucoside, salicin, Ingram, 1. (1960). Studies on benzoate-resistant yeasts. Acta DL-lactate, succinate, citrate, inositol, D-glucosamine, Microbiol7, 95-105. methanol, xylitol or potassium nitrate. Growth occurs James, S.A., Collins, M. D. & Roberts, 1. N. (1994). Genetic in the presence of 50% glucose. No growth occurs in interrelationship among species of the genus Zygosaccharo- the presence of 0-01YO cycloheximide or 1 YO acetic myces as revealed by small-subunit rRNA gene sequences. acid or in vitamin-free medium. Cultures of the type Yeast 10, 871-881. strain, NCYC D2627T, have been deposited in the James, S.A., Collins, M. D. & Roberts, 1. N. (1996). Use of an National Collection of Yeast Cultures, Norwich, UK. rRNA internal transcribed spacer region to distinguish phylo- genetically closely related species of the genera Zygo- saccharomyces and Torulaspora. Int J Syst Bacteriol 46, ACKNOWLEDGEMENTS 189-194. James, S.A., Cai, J., Roberts, 1. N. & Collins, M. D. (1997). A We gratefully acknowledge the assistance of Dr James phylogenetic analysis of the genus Saccharomyces based on 18s Barnett, School of Biological Sciences, University of East rRNA gene sequences : description of Saccharomyces kunashi- Anglia, and Linda Barnett in the preparation of photo- rensis sp. nov. and Saccharomyces martiniae sp. nov. Int J Syst micrographs of the sporulating yeast and Norman Wright at Bacteriol47, 453-460. Unilever Research, Colworth House, and Dr Walter Gams at the Centraalbureau voor Schimmelcultures, Delft, for James, S.A., Roberts, 1. N. & Collins, M. D. (1998). Phylogenetic their kind assistance with the Latin nomenclature of this heterogeneity of the genus Williopsis as revealed by 18s rRNA organism. gene sequences. Int J Syst Bacteriol48, 591-596. Kurtzman, C. P. (1990). DNA relatedness among species of the genus Zygosaccharomyces. Yeast 6, 2 13-2 19. REFERENCES Lemontt, J. F. & MacKay, V. L. (1977). A pleiotropic mutant of Barnett, J. A., Payne, R. W. & Yarrow, D. (1990). Yeasts: yeast expressing the mating specific ‘ schmoo ’ morphology Characteristics and Identification, 2nd edn. Cambridge : during vegetative growth in the absence of exogenous mating Cambridge University Press. hormone. Genetics 86, s38. Cai, J., Roberts, 1. N. & Collins, M. D. (1996). Phylogenetic Mankin, A. S., Skryabin, K. G. & Rubtsov, P. M. (1986). Identi- relationships among members of the ascomycetous yeast genera fication of ten additional nucleotides in the primary structure of Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces yeast 18s rRNA. Gene 44, 143-145. deduced by small-subunit rRNA gene sequences. Int J Syst Munitis, M. T., Cabrera, E. & Rodriguez-Navarro, A. (1976). An Bacteriol46, 542-549. obligate osmophilic yeast from honey. Appl Environ Microbiol Davenport, R. R. (1975). The distribution of yeasts and yeast-like 32, 320-323. organisms in an English vineyard. PhD thesis, University of Neefs, 1. M., Van der Peer, Y., De Rijk, P., Goris, A. & De Wachter, Bristol. R. (1991). Compilation of small ribosomal subunit RNA Davenport, R. R. (1981). Yeasts and yeast-like organisms. In sequences. Nucleic Acids Res 19 (Suppl.), 1987-201 5. Smith’s Introduction to Industrial Mycology, 7th edn. Edited by Neves, L., Pampulha, M. E. & Loureiro-Dias, M. C. (1994). A. H. S. Onions, D. Allsopp & H. 0. W. Eggins. London: Resistance of food spoilage yeasts to sorbic acid. Lett Appl Edward Arnold. Microbiol19, 8-1 1. Davenport, R. R. (1996). Forensic microbiology for soft drinks Pitt, 1. 1. (1974). Resistance of some food spoilage yeasts to business. Soft Drinks Manag Int April 1996, 3435. preservatives. Food Techno1 Aust 6, 238-241. Drubin, D. G. (1991). Development of cell polarity in budding Pitt, 1.1. & Hocking, A. D. (1985). Fungi and Food Spoilage. yeast. Cell 65, 1093-1096. Sydney : Academic Press. Farris, G. A., Fatichenti, F. & Deiana, P. (1981). Microbiological Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new characterization of Sardinian honeys. Tecnol Aliment 4, 18-23. method for reconstructing phylogenetic trees. Mol Biol Evol4, Felsenstein, J. (1985). Confidence limits on phylogenies : an 406-425. approach using the bootstrap. Evolution 39, 783-791. Sand, F. E. M. 1. & van Grimsven, A. M. (1976). Comparison Felsenstein, 1. (1 993). PHYLIP : phylogenetic inference package, between the yeast flora of Middle Eastern and Western version 3.5. University of Washington, Seattle, WA, USA. European soft drinks. Antonie Leeuwenhoek 42, 523-532. Feng, D. F. & Doolittle, R. F. (1987). Progressive sequence Scarr, M. P. & Rose, D. (1966). Study of osmophilic yeasts alignment as a prerequisite to correct phylogenetic trees. J Mol producing invertase. J Gen Microbiol45, 9-1 6. Evol25, 351-360. Thomas, D. S. & Davenport, R. R. (1985). Zygosaccharomyces Genetics Computer Group (1991). Program Manual for the GCG bailii : a profile of characteristics and spoilage activities. Food Package, version 7. Madison, WI : Genetics Computer Group. Microbiol2, 157-169. Grinbaum, A., Ashkenazi, I., Treister, G., Goldschmied-Requven, Tilbury, R. H. (1980). Xerotolerant yeasts at high sugar con- A. & Block, C. 5. (1994). Exploding bottles: eye injury due to centrations. In Microbial Growth and Survival in Extremes of yeast fermentation of an uncarbonated soft drink. Br J Environment, pp. 103-128. Edited by G. W. Gould & J. E. L. Ophthalmol78, 883. Corry. London: Academic Press. Hammond, S. M. & Carr, 1. G. (1976). The antimicrobial activity van der Walt, J. P. & Johannsen, E. (1975). The genus Torulaspora of SO,-with particular reference to fermented and non- Lindner. CSIR Res Rep 325, 1-23. fermented fruit juices. In Inhibition and Inactivation of Veg- van der Walt, 1. P. & Yarrow, D. (1984). Methods for isolation,

326 International Journal of Systernatic Bacteriology 49 Zygosaccharomyces lentus sp. nov.

maintenance, classification of yeasts. In The Yeasts - a Taxo- Watth, A. D. (1989). Relationships among cell size, membrane nomic Study, 3rd edn, pp. 45-104. Edited by N. J. W. Kreger- permeability, and preservative resistance in yeast species. Appl van Rij. Amsterdam : Elsevier. Environ Microbiol55, 2995-2999. Warth, A. D. (1977). Mechanism of resistance of Saccharomyces Yarrow, D. (1984). Zygosaccharomyces Barker. In The bailii to benzoic, sorbic and other weak acids used as food Yeasts - a Taxonomic Study, 3rd edn, pp. 449465. Edited by preservatives. J Appl Bacteriol43, 21 5-230. N. J. W. Kreger-van Rij. Amsterdam: Elsevier.

International Journal of Systematic Bacteriology 49 327